In this application the term “protein of therapeutic value” means pharmacologically active protein, suitable for use in therapy and produced through genetic engineering, including mammalian antibodies, blood product substitutes, vaccines, hormones, cytokines. Potential therapeutic proteins are different in a number of important aspects from classical new drug entities, which are generally low-molecular-weight biologically active compounds. To be efficacious, protein drugs are used in concentration and circumstances which differ markedly from their native counterparts and this may lead to undesirable effects in vivo. To avoid or minimise toxicity and increase efficacy both the physicochemical and biological properties of proteins are being altered generally by some form of protein modification e.g., covalent conjugation with other macromolecules, antibody binding, mutagenesis and glycosylation.
The term “biologically active protein” means the protein molecule which exhibit the same detectable biological activity as the respective naturally existing protein.
The term “monomer unit” means a single polypeptide chain of naturally occurring biologically active protein.
The term “homo-multimer” means a linear polypeptide chain consisting of more than one identical biologically active polypeptide subunits, which are connected to each other via peptide linker molecule in a such manner that connection of polypeptide subunit through disulfide bonds is excluded.
The term “genetically fused” means that homo-multimer protein is obtained using recombinant DNA methodology: construction of artificial DNA fragment consisting of two or more connected genes (coding sequences) of monomer proteins via linker DNA sequence, introduction of the DNA fragment into the vector and expression of the protein in the selected cell type. The recombinant protein consists from monomer proteins connected with linker peptide sequences (protein could be isolated after applying special purification procedure) in contrast with “chemically conjugated” protein that is obtained by chemical joining of two or more monomer proteins using specific chemical methods and “chemically modified” protein that is obtained by chemical modification of monomer protein using chemical agents or polymer residues.
Advances in biochemistry, protein chemistry and molecular biology over the last twenty-five years have spurred the increased use and development of recombinant proteins as injectable therapeutic agents. Protein and peptide biopharmaceuticals have been successfully used as very efficient drugs in therapy of many pathophysiological states since the first recombinant product insulin was approved in 1982, One group of approved first generation protein biopharmaceuticals mimics native proteins and serves as replacement therapy, while another group represents monoclonal antibodies for antagonist therapy or activating malfunctioning body proteins [Jev{hacek over (s)}evar S. et al., PEGylation of therapeutic proteins, Biotechnol. J., 5, 113-128 (2010)]. The main drawbacks of the first-generation biopharmaceuticals are their suboptimal physicochemical, pharmacokinetic and pharmacodynamic (PK/PD) properties. Main limitations are physicochemical instability, limited solubility, proteolytic instability, relatively short elimination half-life, immunogenicity and toxicity. Consequently, protein therapeutics are mainly administrated parenterally [Jev{hacek over (s)}evar S. et al., Biotechnol. J., 5, 113-128 (2010)].
Wide range of technologies have been developed during the last decade directed to the development of second-generation biopharmaceuticals encompassing the main products of first-generation protein drugs by conferring to them improved PK/PD characteristics. Extension of in vivo circulating half-life time is the main goal of such therapeutics. Long-acting forms of erythropoietin (EPO, containing two N-linked oligosaccharide chains, under the name of Aranesp), granulocyte colony-stimulating factor (pegylated-G-CSF under the name of Neulasta) and interferons (pegylated IFN alfa-2b under the name of PEG-Intron or pegylated-IFN alfa-2a under the name of Pegasys) are successful examples of second-generation biopharmaceuticals which gained the status of blockbuster drugs.
One of widely used method to prolong plasma half-life time of the target therapeutic protein is chemical modification focusing on increasing the size of therapeutic protein by conjugation with natural or synthetic polymers using well established procedures known as PEGylation (chemical conjugation with polyethylene glycol, Veronese F. M. et al., Protein PEGylation, basic science and biological application in PEGylated Protein Drugs: Basic Science and Clinical Applications, ed. F. M. Veronese, 11-31(2009), polysialylation (chemical conjugation with polysialic acid, Sanjay Jain et al. Polysialylation: The natural way to improve the stability and pharmacokinetics of protein and peptide drugs. dds&s Vol 4 No 1 May (2004), HESylation (chemical conjugation with hydroxyethylstarch, International Patent Application WO 02/080979 and International Patent Application WO 03/000738) and others.
Another method of modification of clearance of therapeutic proteins is through chemical cross-linking or genetic modification to fuse proteins of interest with long-living plasma proteins like albumin, immunoglobulin, or portions of these proteins (Sheffield, Modification of clearance of therapeutic and potentially therapeutic proteins, Curr. Drug Targets Cardiovasc Haematol Disord., 1,1-22, (2001).
Modification of N- and C-terminus of a therapeutic protein or replacement of amino acids which are known to be susceptible for enzymatic cleavage is also used as a strategy to reduce immunogenicity and proteolytic instability and therefore to improve plasma half-life time (Werle M and Bernkop-Schmurrch, Amino acids, 30, 351-367 (2006).
However, all these modifications often cause significant reduction of the biological activity of the protein of interest or elicit antibody formation. Due to this a relatively limited range of proteins with improved plasma half-life time are in clinical use. Therefore, search of new means for alteration of circulation half-life of therapeutic protein remains a challenging task of current biotechnology.
Patent U.S. Pat. No. 5,580,853 describes methods of preparing multimeric erythropoietin derivatives comprising two or more erythropoietin molecules covalently linked together by one or more thioether bond(s). These erythropoietin multimers exhibit increased biological activity and prolonged circulation time. In this method a first erythropoietin derivative was produced by chemically reacting wild type erythropoietin with a hetero-bifunctional cross-linking reagent containing a cleavable disulfide bond group. The disulfide bond was reduced to produce erythropoietin containing a free sulfhydryl group. A second erythropoietin derivative was produced by reacting wild type erythropoietin with a hetero-bifunctional cross-linking reagent containing a maleimido group. The first and second erythropoietin derivatives were reacted together, thereby forming at least one thioether bond between the sulfhydryl and maleimido groups, thus forming a homodimer or homotrimer of erythropoietin. These multimeric erythropoietin molecules exhibit biological activity comparable to wild type erythropoietin and prolonged circulating half-life in vivo, relative to wild type erythropoietin. However, chemical conjugation of protein molecules, e.g., erythropoietin into multimeric form may be accompanied by non-specific chemical modification via functional group of amino acid residue participating in receptor binding. This could decrease the biological activity of the multimeric product. In general, chemical modification may generate unfavourably linked products which must be separated from the correctly linked target product and other by-products. Such modification process is expensive and requires additional purification process steps trying to obtain desired product with reproducible activity and quality characteristics.
EP1334127 discloses single-chain multimeric polypeptides comprising at least two units of a monomeric polypeptide covalently linked via a peptide bond or a peptide linker. Monomeric polypeptide belongs to the protein type that is biologically active in monomeric form. Here, at least one monomeric unit of the construct differs from the corresponding wild-type monomeric polypeptide by at least one added or removed amino acid residue (Lys, Cys, Asp, Glu or His) which serves as an attachment site for further chemical conjugation with non-polypeptide type moiety. The polypeptide is preferably a G-CSF dimer with attached polymer molecule, preferably polyethylene glycol molecules. This patent does not provide any information on the impact of peptide linker on the biological activity of multimeric peptides.
More attractive approach related to production of biologically active multimeric protein derivatives is described in U.S. Pat. No. 5,705,484 that discloses biologically active multimeric polypeptide molecule in which two or more monomeric subunits are linked together as a single polypeptide and prepared as genetically fused multimer. The fusion multimers specifically include PDGF fusion dimers in which protein monomer units are linked via spacer moiety selected from the pre-pro region of a PDGF precursor protein. These fusion multimers are more easily and rapidly refolded than unfused multimers and eliminate the simultaneous formation of undesirable polypeptide by-products during refolding. However, the use of pre-pro sequence as a linker is not favourable due to possible digestion by proteases which commonly participated in the processing of pre-pro domains of a protein molecule.
WO 01/03737 discloses fusion proteins comprising a cytokine or growth factor fused to an immunoglobulin domain, in particular IgG. Also disclosed are multimeric fusion proteins comprising two or more members of the growth hormone superfamily joined with or without a peptide linker. The peptide linker is SerGly, Ser(GlyGlySer)n, wherein n is 1 to 7 or sequences Ser(GlyGlySer) or Ser(GlyGlySer)2. However examples are only provided for fusions with IgG which are regarded as hetero-multimeric fusion proteins.
For separation domains of a bifunctional fusion proteins several lengths of helix-forming peptides linkers with sequence A(EAAAAK)nA (n=2-5) were introduced (R. Arai et all., Prot. Eng., 14 529-532 (2001) showing that such linkers allow controlling the distance and reduce the interference between the domains. This approach is used for separation of different domains of the same type of proteins.
WO2005/034877 describes a polypeptide comprising a G-CSF domain linked to a transferrin domain through Leu-Glu linker. Such fusion can be used for treating various diseases as orally delivered pharmaceutical, but transferrin domain activity is very poor.
Patent U.S. Pat. No. 7,943,733 discloses the role of linker introduced between the partners of hetero-multimers of transferrin-based fusion proteins demonstrating that the insertion of alpha-helical linker between two fusion partners increase the expression level of the fusion. LEA(EAAAK)4ALE sequence between the partner of the fusion comprising a carrier protein (transferrin, serum albumin, antibody and sFV) and therapeutic protein (G-CSF, an interferon, a cytokine, a hormone, a lymphokine, an interleukin, a hematopoietic growth factor and a toxin). However, this patent is dedicated for the production of hetero-fusion construct where protein partners are different protein molecules, representing different activities.
A program to generate all possible linker sequences for fusion proteins has been recently published (C. J. Crasto and J. a. Feng, Prot. Eng., 13, 309-312 (2000). It serves as a tool for rational design of desired linker sequence and its length. However this program could not predict the biologic activity of the final fusion proteins due to complexity of the factors enabling the active structure of the fusion proteins. So, only experimental testing of the different selected linker structures is capable to give the evidence of protein activity.
Thus, among the existing methods for production of long-acting biopharmaceuticals a method of linking monomer unit of the protein of interest into multimers is less time-consuming and allowing to protect multimer construction from the lost of biological activity. However, known method of chemical conjugation of protein monomer into homo-multimers albeit protected biological activity and prolonged circulating half-life did not assure reproducibility of multimer quality parameters.
Method for production of single-chain multimeric polypeptides by introduction into one monomeric unit of amino acid residue for subsequent attachment of PEG in principle mimics well-known methodology of therapeutic protein PEGylation with the only difference that dimer form of the protein instead of monomer is used.
With regards to this, the present invention proposes a method for production of biopharmaceuticals with increased circulation half-life, using genetic fusion of protein monomer units into homo-multimer construct via linkers of defined length. Sequence and structure of the linker is designed to achieve accessibility of each monomer unit to interact with specific receptor of selected monomer of therapeutic protein. Owing to this, no further attachment of polymer, e.g. PEG molecules is required.
Therefore, it is an object of the present invention to provide a multimeric form of therapeutic proteins of interest having biologically active monomer units and improved PK/PD characteristics.
It is a further object of the present invention to provide a multimeric form of therapeutic proteins that can be produced via recombinant DNA technology without formation of undesirable dimer and oligomer forms cross-linked via disulfide bonds.
When molecular mass of the recombinant protein is increased the technologic problems frequently multiply during preparation of the target protein to the purity level in compliance with the requirements for pharmaceutical substance. On the other side the increase of molecular mass of the target protein is associated with the risk of increased propensity for aggregation especially when high expression level of the protein leads to its accumulation in insoluble form (inclusion bodies). In this case technology approaches applied for preparation of monomer protein are no more suitable for isolation and purification of multimeric proteins. Here serious attention should be paid for the proper selection not only of each process stage but also for the compatibility of whole process steps assuring as high as possible biologic activity of the protein with its increased molecular mass at the end of purification cycle. So, this proves the necessity of a new approaches to the preparation of multimeric proteins.